Discharge properties of motoneurons supplying distal forelimb muscles in the cat

192

Brain Research 37911980 J I t)':2-195

Elsevier BRE 21680

Discharge properties of m~tonotmm~ ~ ~ ¢ l l t a t

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B.R. BOTTERMAN and F.C. COPE Department o f Cell Biology and Anatomy, University of Texas Health Science Center at Dallas. Dallas. FX 75235 (U.S.A. r

(Accepted April 8th. 1986 Key words: spinal cord

cervical motoneuron

electrical properties

distal forelimb m cat

Discharge properties of cat cervical motoneurons innervating distal forelimb muscles were investigated by inttacellular current injection. Values for rbeobase current, afterhyperpolarization duration and several measures of repetitive dischargecharacteristics were in most respects similar to those obtained for hindlimb motoneurons.

Most of what is known a b o u t the excitability of a m o t o n e u r o n s has been o b t a i n e d from studies of those supplying muscles in the cat hindlimb. These motoneurons vary systematically in excitability m e a s u r e d as rheobase current 4'1~'13 and in the relation between repetitive discharge frequency and injected current ( F - I relation) l'7"8"1°'H. The extent to which hindlimb m o t o n e u r o n s represent spinal a - m o t o n e u r o n s in general is largely u n d e t e r m i n e d , however, owing perhaps to the difficulty in obtaining stable intracellular records from o t h e r regions of the spinal cord. Indeed. there are reasons to expect m o t o n e u r o n s belonging to various m o t o r pools differ electrically. Cranial m o t o n e u r o n s supplying the unusually fast contracting muscle units of lateral rectus 5 exhibit F - I relations 6 that are much s t e e p e r than hindlimb motoneurons s. A d d i t i o n a l l y , recent investigations in this l a b o r a t o r y 3 show that fast-twitch forelimb m o t o r units require substantially higher rates of activation to produce the same relative p e r c e n t a g e of m a x i m u m tetanic tension as h i n d | i m b units. F r o m this observation we hypothesized that forelimb m o t o n e u r o n s might be specialized, as s o m e cranial m o t o n e u r o n s a p p e a r to be 6. so as to optimally utilize the distinctive mechanical p r o p e r t i e s of their muscle units In this report, and earlier in abstract form 2. we present what is to our knowledge the most extensive study available on active electrical properties of spinal moto-

neurons other than those supplying hindlimb muscles. Intracellular records of m o t o n e u r o n m e m b r a n e potential were m a d e in 6 adult cats deeply anesthetized with p e n t o b a r b i t a l ( N e m b u t a l . 35 mg/kg). The cervical m o t o n e u r o n s targeted for study were antidromically activated by supramaximat electrical stimulation (0.01 ms pulses r e p e a t e d at 0.5 pps) through bipolar electrodes positioned in the left forelimb. Stimulated nerves included the ulnar ( U L N ) nerve and the m e d i a n ( M E D ) nerve together with two of its c o m p o n e n t s , n a m e l y the nerves supplying the palmaris longus ( P A L ) and flexor carp1 radiatis ( F C R ) muscles. M o t o n e u r o n s activated in this way were p e n e t r a t e d in cervical spinal segments C 8 and caudal C, using glass microptpettes filled with 2 M potassium citrate and having inpu~ resistances ranging from 5 to 15 Mr2. The action potential was the sole criterion for the quality o f intracellular penetration. since rectification of the microelectrode caused by long-duration current injection (see below) prevented reliable m e a s u r e m e n t of m e m b r a n e p o t e n tial. Intracellular records were accepted for analysis for so long as antidromic action potentials, checked intermittently during recording, e x c e e d e d 70 mV in amplitude. This qualifying level is at least as high as that which has been used in studies making the same electrical m e a s u r e m e n t s in hindlimb rnotoneurons4.7.1 ~.

Correspondence: B.R Botterman. Department of Cell Biology and Anatomy, University of Texas Health Science Center at Dallas. 5323 Harry Hines Blvd.. Dallas. TX 75235. U.S.A.

0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. ( Biomedical Division I

193

All measurements involved monitoring the response of m o t o n e u r o n m e m b r a n e potential to various durations and intensities of depolarizing current pulses injected through the micropipette. (It should be noted that dorsal roots T 1 through C 6 were cut to remove the potential contribution of synaptic current from primary afferent fibers.) R h e o b a s e current (Irh) was m e a s u r e d on-line as the minimum amount of current, delivered in 50 ms square pulses at 0.5 pps, required to achieve m o t o n e u r o n discharge threshold. Next, long current pulses (2.5 s) were injected at various intensities that p r o d u c e d repetitive discharge in motoneurons. The discharge rates were m e a s u r e d off-line from A C - c o u p l e d records (10 H z - 5 kHz) of m e m b r a n e potential stored on F M tape ( 0 - 5 kHz). Action potential spikes were converted to T T L pulses via a window discriminator, and used to trigger interrupt circuits on an LSI-11/23 computer. The timing and pattern of the spike train was written into m e m o r y at 0.1 ms resolution, from which discharge rates and interspike intervals were m e a s u r e d and stored. This analysis provided for the description of the relation between m o t o n e u r o n discharge frequency and current intensity, i.e. the F - I relation. Finally, brief (0.1 ms) current pulses were injected to elicit single action potentials at 0.5 pps. D C - c o u p l e d records ( 0 - 5 kHz) of these potentials were stored on F M tape and later digitized at 0.1 ms/pt by a Nicolet 1174 signal averaging computer. Digitized records of 8 potentials were averaged and afterhyperpolarization ( A H P ) duration measured from action potential onset to the return of m e m b r a n e potential from hyperpolarized levels to baseline. We m e a s u r e d A H P duration from cervical m o t o n e u r o n s to test whether it is related to limits in firing rate as it is for hindlimb m o t o n e u r o n s 9. F o r e l i m b m o t o n e u r o n s were capable of firing continuously in response to long-lasting injected current. It was our impression that all m o t o n e u r o n s encountered (n = 26) were capable of discharging throughout the 2.5 s current pulse, yet damage p r e s u m a b l y caused by the microelectrode often p r e v e n t e d detailed study of this behavior. We noted that short duration bursts of spikes were observed for some cells which had previously fired continuously at the same current strength and that this bursting activity coincided with deterioration of the cell as judged by a decrease in amplitude and often an increase in duration

of the action potential. W e found no evidence for phasic discharge behavior on the part of u n d a m a g e d distal forelimb motoneurons. The F - I relation was characterized from four or more different current strengths in 11 motoneurons. W e base the reliability of these data on the following observations. First, these 11 cells fired continuously throughout the 2.5 s current pulse and exhibited no signs of deterioration in records accepted for analysis. Second, the linear regression coefficients calculated from steady state firing rate (see below) vs current strength were in all cases significant at P < 0.025 (one-tailed t-test). This demonstrates that firing rate increased in a predictable way with current strength. Finally, firing rate was reasonably well r e p r o d u c e d whenever recording conditions permitted repeated measures of repetitive discharge at the same current strength: the mean difference in steady-state firing rate for 17 cases where two trials were made at the same current strength was 5.2 +_ 3.9 pps (S.D.). Fig. 1 displays for an U L N m o t o n e u r o n the F - I relation that was typical of the 11 cells just mentioned. Comparison of instantaneous firing frequency across the first, second and third interspike intervals and at intervals 1 s later d e m o n s t r a t e d adaptation in firing rate. It can be seen particularly for the greatest current strengths that firing rate declines progressively over successive interspike intervals. This adaptation was most p r o n o u n c e d over the initial 4 - 5 spikes, af-

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Fig. 1. Relationship between the instantaneous discharge frequency and current intensity of an ulnar motoneuron. Firing rate was measured for the first (1), second (2) and third (3) interspike intervals of the spike train elicited by a 2.5 s depolarizing current pulse. Steady-state (S) firing rate was measured 1 s after the current pulse was delivered to the motoneuron. The steady-state slope for this motoneuron was 1.1,)2imp/s/nA. Motoneuron spike amplitude was 87 mV at resting membrane potential.

194 ter which firing rate declined much more gradually.

{n -- 26) that included those in Table I. rheobase current ranged from 2 to 16 nA. The average for this

A measure of late adaptation, expressed as the percent decrease in F - I slope at 2.5 s relative to 1.0 s af-

sample was 7.4 ___4.3 n A IS.D.I with a median score

ter current onset, ranged from - 5 to - 2 2 % . Fig. 1 also shows that early in the spike train firing

of 8 nA. A H P duration was measured for 19 of these cells. It ranged from 43 to 135 ms. averaged 78 -- 23

rate did not increase linearly with current strength.

ms (S.D.) and had a median score of 78 ms. There was a tendency for m o t o n e u r o n s with the Longest

The regions of relatively slow and rapid increment in rate with current have been designated as primary and secondary range firing, respectively s. While sec-

A H P durations to have low rheobase values In = 14: Pearson correlation coefficient, r ---: -0.64: t' < I/.01 using one-tailed t-test 1.

ondary range firing was observed in most cases for

for this purpose). This precluded determination of m a x i m u m primary range firing rate, but the maxi-

In most regards the data just presented were quite similar to those reported for hindtimb m o t o n e u r o n s . M i n i m u m and m a x i m u m steady-state firing rates for hindlimb m o t o n e u r o n s (15 and 54 pps on average, respectively7'9) are comparable to those that we found

m u m 'steady-state' rates (taken at 1 s from spike train

for the forelimb. Furthermore. these m i n i m u m and

onset) that were observed are presented in Table 1. Average m a x i m u m steady state rate was 44.3 ~ 17

maximum rates for fore- and hindlimb m o t o n e u r o n s were obtained over similar current strengths. With regard to the average a m o u n t of injected current required to reach threshold for forelimb m o t o n e u r o n s .

the first and second interspike intervals, it was not evident for intervals later in the train (possibly because the current strengths used were not sufficient

pps (S.D.). The absolute m a x i m u m instantaneous frequency, which was always obtained from the first

i.e rheobase current, our values tell within the range reported for the hindlimb4'~3. It appears then that the electrical excitability of distal forelimb m o t o n e u r o n s is well represented by what has been described for hindlimb motoneurons. One might speculate from

interspike interval, ranged from 303 to 500 pps. M i n i m u m repetitive firing rates and their associated current levels are also listed in Table I. The m i n i m u m a m o u n t of current required to attain repetitive discharge threshold, expressed in multiples of

this that specializations of cervical m o t o n e u r o n s are not necessary to meet the requirements of distal forelimb motor units for faster activation rates to produce

I~h, averaged 3.6 + 1.8 n A (S.D.; range 1.4-6.3 x Irh), The m e a n rate at these current strengths was 18.1 + 5.2 pps (S.D.). For a larger sample of distal forelimb m o t o n e u r o n s

the same a m o u n t of isometric tension as hindlimb

TABLE I Electrical and discharge properties of distal forelimb motoneurons

Max. steady-state rate is the observed maximum steady-state rate and may be somewhat less than the maximum primary range rate s since secondary range firing was not observed under steady,state conditions. AP = antidromic action potential. Motoneuron species

Current range (hA)

Steady-state slope (imp/s/nA)

Max. steadystate rate (pps)

Min. steadystate rate (pps)

Min. stead),state current (nA)

Rheobase current (hA)

,4 [1P duration ~ms)

A i' amplitude (mV)

MED MED MED MED MED MED FCR PAL ULN ULN ULN

20-45 10-25 18-35 30-60 13-50 10-46 5-15 14-35 10-45 15-30 7-25

1.00 2.02 1.47 0.98 2.16 0.92 1.28 0.75 1.02 0.82 1.84

43 43 45 53 91 47 25 30 42 36 32

20 16 22 NM 24 19 12 16 13 27 12

20 8 18 NM 13 10 5 14 10 15 7

16 3 8 10 10 3 2 8 7 8 4

77 100 84 80 82 NMa 121 69 78 88 t35

83 8~ 87 73 94 91 77 75 87 82 t01

a Depolarizing pulses consistently elicited two pulses, precluding measurement of AHP duration. NM = no measurement.

195 units 3. D i r e c t c o m p a r i s o n s of the m o t o n e u r o n and

age m u l t i p l e of r h e o b a s e c u r r e n t for h i n d l i m b m o t o -

muscle unit c o m p o n e n t s of single m o t o r units m u s t be

n e u r o n s ( a v e r a g e 1.5) 7. Finally, the direct r e l a t i o n

m a d e to test this hypothesis. A m o n g the differences f o u n d b e t w e e n fore- and

b e t w e e n m i n i m u m and m a x i m u m steady-state firing

hindlimb m o t o n e u r o n s , the slope of the steady-state

limb m o t o n e u r o n s 9 was n o t e v i d e n t in our sample.

rate with A H P d u r a t i o n that has b e e n f o u n d for hind-

F - I relation t e n d e d to be larger for h i n d l i m b m o t o -

F u r t h e r study of f o r e l i m b m o t o n e u r o n s will r e v e a l

n e u r o n s (range 0 . 4 - 4 . 5 imp/s/nA7; cf. T a b l e I). A l s o ,

w h e t h e r t h e s e a p p a r e n t d i f f e r e n c e s are significant.

r e p e t i t i v e firing t h r e s h o l d o c c u r r e d at a l o w e r aver-

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motoneurones stimulated by long-lasting injected currents, Acta Physiol. Scand., 65 (1965) 65-73. 8 Kernell, D., High-frequency repetitive firing of cat lumbosacral motoneurones stimulated by long-lasting injected currents, Aeta Physiol. Scand., 65 (1965)74-86. 9 Kernell, D., The limits of firing frequency in cat lumbosacral motoneurones possessing different time course of afterhyperpolarization, Acta Physiol. Scand,, 65 (1965) 87-100. 10 Kernell, D., Input resistance, electrical excitability, and size of ventral horn cells in cat spinal cord, Science, 152 (1966) 1637-1640. 11 Kernell, D., Rhythmic properties of spinal motoneurones innervating muscle fibres of different speed in m. gastrocnemius medialis of the cat, Brain Research, 160 (1979) 159-162. 12 Sypert, G.W. and Munson, J.B,, Basis of segmental motor control: motoneuron size and motor unit type? Neurosurgery, 8 (1981) 608-621. 13 Ulfhake, B. and Kellerth, J.-O., Electrophysiological and morphological measurements in cat gastrocnemius and soleus a-motoneurones, Brain Research, 307 (1984) 167-179.